PROJECT SUMMARY/ABSTRACT Exposure to environmental agents including heavy metals, air pollutants, pesticides and radiation induces the formation of aberrant RNA-DNA hybrids termed R-loops. Unresolved R- loops are the cause of DNA damage and genome instability, and are also seen in neurodegenerative diseases, e.g. amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), fragile X syndrome, and Friedreich's ataxia. The elimination of these pathological RNA- DNA hybrids is dependent on the Fanconi anemia (FA) pathway of DNA damage response and on the tumor suppressors BRCA1 and BRCA2. Recent studies have also implicated several nucleic acid motor proteins, namely, FANCM, Senataxin (SETX), and Aquarius (AQR), in R-loop resolution. AQR is the least understood among these motor proteins and the mechanism by which AQR helps resolve R-loops remains elusive. We hypothesize that AQR engages and dissociates R-loop structures to prevent their accumulation in cells. In this project, we will apply our considerable expertise in molecular studies of nucleic acid motor proteins to define the mechanism by which AQR functions in the R-loop resolution and genome preservation. In Aim 1, we will purify AQR, investigate its association with R-loop structures, and test its ability to dissociate these structures. Mutants of AQR defective in nucleic acid binding or ATP hydrolysis will be generated to establish the relevance of these attributes in vitro and in cells. In Aim 2, we will identify cofactors of AQR, purify them, and define their influence on AQR's activity in R-loop dissociation in reconstituted biochemical systems and in cells. The results from our project will shed light on a poorly understood pathway of genome preservation and are expected to contribute toward the development of novel strategies to avoid the accumulation of R-loops upon exposure to environmental stress and mutagens, and to treat human diseases including neurodegeneration and cancer.